Device and method for processing biological material

ABSTRACT

The invention relates to a device for processing biological material, which at least comprises a chamber which at least can be closed in relation to the outside and which comprises an inner space for receiving said biological material, wherein said chamber comprises at least one electrode which is placed in contact with said inner space of said chamber and is provided for generating an electric field. The invention also relates to a method for processing biological material, wherein said biological material is introduced into the inner space of a chamber which at least can be closed in relation to the outside and which comprises at least one electrode which is placed in contact with said inner space of said chamber and is provided for generating an electric field which is generated in said inner space after introducing said biological material by applying voltage to said electrode and a further electrode which is in contact with said inner space of said chamber. According to the invention, said chamber comprises at least one inlet line which comprises at least one opening arranged close to said electrode. According to the inventive method, said biological material is almost completely rinsed out of said inner space of said chamber by means of a solution after said electric field is generated, said solution being guided via an inlet line of said chamber along at least one electrode.

TECHNICAL FIELD

The invention relates to a device for treating biological material,which at least comprises a chamber which at least can be closed inrelation to the outside and which comprises an inner space for receivingsaid biological material, wherein said chamber comprises at least oneelectrode which is placed in contact with said inner space of saidchamber and is provided for generating an electric field. The inventionfurther relates to a method for treating biological material, whereinsaid biological material is introduced into the inner space of a chamberwhich at least can be closed in relation to the outside and whichcomprises at least one electrode which is placed in contact with saidinner space of said chamber and is provided for generating an electricfield which is generated in said inner space after introducing saidbiological material by applying voltage to said electrode and a furtherelectrode which is in contact with said inner space of said chamber.

PRIOR ART

Transferring biologically active molecules, such as, for example, DNAs,RNAs or proteins, into living cells is an important tool for analysis ofbiological functions of these molecules. Electroporation is a preferredmethod for transferring foreign molecules into the cells, which incontrast to chemical methods does not depend on a simultaneous transportof other biologically active molecules. During electroporation theforeign molecules are introduced into the cells from a buffer solutionadapted to the cells or a cell culture medium by a short-time currentflow, whereby the cell membrane is made permeable for the foreignmolecules by effect of the short electric pulses. The cell suspension isusually provided in a so-called cuvette, i.e. a small container which isopen at the top and which comprises two parallel and oppositely arrangedelectrodes disposed in the sidewalls near the bottom and serving for theapplication of electric voltage. Through the temporarily emerging“pores” in the cell membrane the biologically active molecules initiallyreach the cytoplasm where they eventually already exert their functionto be analysed. At certain conditions the molecules subsequently alsoenter the nucleus of the cell.

Due to the temporarily applied intensive electric field, i.e. a shortpulse with high current density, cells, derivatives of cells,subcellular particles and/or vesicles can also be fused. During thisso-called electrofusion at first, for instance, the membranes of thecells are brought in close contact by an inhomogenous alternatingelectric field. The subsequent application of an electric field pulseleads to an interaction of parts of the membranes finally leading tocell fusion. Comparable devices such as the ones used forelectroporation can be used for electrofusion as well.

Containers as mentioned above are known and primarily used forelectroporation or electrofusion in the form of cuvettes having insertedelectrodes made of metal. Containers used for this purpose are mostlysmall vessels which are closed at the bottom and open at the top andwhose inner space is built by two pairs of parallel and oppositelyarranged sidewalls. The inner space serves for receiving of the cellsuspension, i.e. usually an aqueous buffer solution or a cell culturemedium, in which the cells to be treated are suspended. Such cuvettesmostly comprise a pair of electrodes for application of an electricvoltage disposed near the bottom of a pair of oppositely arrangedsidewalls. During an electric discharge an electric current flowsthrough the cell suspension between both electrodes, which enables anintroduction of nucleic acids or other molecules into the cells or,depending on the selected conditions, leads to fusion of cells. Theelectrodes are mostly made of metal, wherein aluminium is frequentlyused.

For example, a chamber for treating cells in an electric field is knownfrom German Patent DE 33 21 239 C2, which has an inner space forreceiving of a suspension comprising living cells, wherein at least twoelectrodes each project into said inner space. As usual, theseelectrodes serve for application of voltage in order to generate anelectric field between said electrodes, wherein the cells are exposed tosaid electric field. Though the chamber is provided for cell fusion, itmay also be used for transferring of nucleic acids into cells, i.e. theso-called electroporation. The inner space of the chamber ishermetically sealed at all sides, wherein one area of the wallsurrounding said inner space can be perforated by a needle or canula.Thus, the wall may partially comprise a foil made of acetyl cellulose,which can be perforated. The cell suspension can be poured in thechamber and removed therefrom through this perforatable foil. This isbeneficial because treating of the cells may occur under asepticconditions. However, after treating the cells, the chamber can only berinsed circumstantially and unsatisfactory so that always a remarkableportion of treated cells remains in the inner space of the chamber.

A chamber for treating cells in an electric field is also known fromGerman Patent DE 33 17 415 C2. With this chamber, the inner space islimited by an inner body and an outer casing which surrounds thelongitudinal axis of the inner body equally distanced. The electrodesprojecting into the inner space surround the inner body in the form of amulti-threaded screw having an equal lead. The chamber has a sealableinlet line for introducing the cell suspension and a sealable outletline for removing the cell suspension. Thus, this chamber may be used asflow through chamber as well. In this case, a predetermined amount ofcell suspension is pressed or sucked into the chamber, electrictreatment is then carried out, and finally the cell suspension locatedin the chamber is pressed or sucked out of the chamber and replaced by anew amount of cell suspension. After electric treatment of the cells,the inner space can be rinsed with a cleaning solution which may bepressed into the inner space via a pore system. However, also in thisembodiment, effective flushing of the chamber after electric treatmentof the cells without the use of a cleaning solution, i.e. underpreservation of the viability of the cells, is not possible as thesolution cannot flow along the entire surface of the electrodes withhigh flow rate.

A further chamber for treating cells in an electric field is known fromGerman Patent DE 35 22 610 C2, wherein the walls building the innerspace for receiving the cell suspension consist of an inner and an outerelectrode. The chamber includes an opening for introducing the cellsuspension, which is closed by a plug, and an outlet opening forremoving treated cells. A defined amount of suspension may be introducedinto the inner space using a pipet and subsequently treatedelectrically. By introducing an additional exactly dosed amount ofsolution the cell suspension is then pressed downward through the outletopening out of the inner space. Due to the geometry of the chamber andthe arrangement of electrodes in the form of an inner and outerelectrode, it is also not feasible to rinse the treated cells with highflow rate.

U.S. Pat. No. 4,849,089 descibes a chamber for electrcally treatingvesicles, which is formed by a circular collar. The inner space forholding the suspension of vesicles is built by an inner ring which isspaced from the collar. The isolating material disposed between thecollar and the inner ring is broken through by two passageways whichrender the inner space accessible from the outside. The suspension maybe filled in or removed from the inner space through these passageways.The electrodes each consist of circular plates which are placed in thecollar from above and the bottom, and which form the bottom plate andthe top cover of the inner space, respectively. Also with this device,due to the circular geometry of the inner space and the electrodessituated at top and bottom an effective flow-through and hence completlyrinsing the treated vesicles is not feasible.

It is a drawback of all devices and methods for electroporation andelectrofusion known by now that the treated cells or vesicles can onlybe removed from the chamber incompletely, i.e. with relatively high lossof biological material. In particular, if the voltage pulses used have avery high field strength, i.e. the electric field has a high currentdensity, cell material often deposits on the electrodes, primarily onthe cathode. Additionally, intense gas formation often occurs leading tofoam formation what also hampers the complete removal of treated cells.

SUMMARY OF THE INVENTION

It is therefore the problem to be solved by the invention to provide adevice and method as initially mentioned, which avoid the drawbacksmentioned above and allow for recovery of treated cells from a chamberwhich is sealed to the outside as complete as possible.

According to the invention the above problem is solved by a devicecomrising a chamber which comprises at least one inlet line comprisingat least one opening arranged close to said electrode. Due to thisspecific arrangement the inner space of said chamber can be effectivelyrinsed, even in a closed state and under aseptic conditions, wherein inparticular the crucial region of the electrode is rinsed by the solutionfirst. Thus, biological material adhering to inner surfaces of thechamber can be effectively removed leading to approximately completerecovery of the material employed. This is primarily beneficial with theuse of valuable material which is only accessible in small amounts.

It is thereby advantageous if said inlet line is designed like a tube,i.e. has a minor cross section in proportion to its longitudinalextension. High flow rates within the inlet line and hence at istopening can be achieved in this configuration.

In an advantageous embodiment of the invention the inner diameter ofsaid inlet line may be decreased in the direction of said electrode.This also leads to a high flow rate at the opening of the inlet line andthus to an effective rinsing operation. The diameter may therebydecrease gradually and continuously over the whole length of the inletline or it may be limited to the region near the opening. In the lattercase, the opening may be designed like a nozzle.

In a further embodiment of the invention it is provided that at leastone reservoir for receiving a solution, which is built of a wall, is atleast connectable to said inner space via said inlet line. This isparticularly advantageous when the cells are not trated in a cellculture medium but in a buffer solution which is optimized forelectrical treatment of cells. In this case, it is necessary to dilutethe buffer solution with a solution which is adapted to the cellsimmediately after the end of the treatment. Due to the fact that thereservoir can be connected directly to the inner space it is ensuredthat the dilution of the buffer solution used during treatment isaccomplished fast and uncomplicated.

The reservoir containing the solution for rinsing the chamber can beconnected to the inner space, wherein said inner space of said chamberand said reservoir may be separated from each other by a separatingunit, wherein said solution can be selectively introduced into saidinner space of said chamber through said separating unit. This isprimarily beneficial if the reservoir is firmly linked to the innerspace since the solution shall not enter the inner space until thetreatment of cells is finished. The separating unit may thereby be avalve or a fragile membrane which can be destroyed by applying pressure.In this embodiment, the chamber may be rinsed by simple manipulationfrom the outside what is primarily important under aseptic conditions.

For clinical applications which are accomplished under asepticconditions it is provided that the chamber is at least asepticallysealed in relation to the outside. If the chamber shall be transportedin the form of a closed unit that is prefilled, for example, with abuffer solution, e.g. comprising solved biologically active molecules,the chamber may additionally be water-proof and/or gas-proof.

In an particularly advantageous embodiment of the invention, the wallbuilding the reservoir may comprise an elastic and/or deformablematerial. Hereby, pressure can be applied from the outside to thesolution situated within the reservoir so as to allow streaming of thesolution into the inlet line at high flow rate. However, if in analternative embodiment the solution is sucked out of the reservoir bynegative pressure, the wall can deflate in proportion to the outpouringsolution.

According to the invention, the reservoir may be at least connectable tothe chamber. For example, said reservoir may be connected to saidchamber building one piece so that both parts can be transported andused as one unit. But it may also be connectable to said chamber via aconnecting member, preferably a Luer lock, so that both parts can betransported and stored separately. In the latter case, variousreservoires which are already present with the user can be used with achamber according to the invention. In an advantageous embodiment of theinvention, said chamber and said reservoir form a unit which is at leastaseptically sealed in relation to the outside.

In a particularly advantageous embodiment of the invention, it isprovided that said chamber comprises at least one wall area which isself-sealing and can be perforated, preferably by a canula, and/or whichis equipped with at least one inlet comprising a connecting member,preferably a Luer lock. It is for instance feasible to introduce asuspension of cells or other biological material into the inner space ofsaid chamber through such wall area or a special connecting member underaseptic conditions.

Recovery of biological material can be further improved if the chamberhas a minor cross-section and/or is formed like a serpent or spiralsince this results in a high flow rate of the solution within the innerspace.

In an alternative embodiment of the invention, the chamber may bedivided in several subunits by at least one dividing member. Thereby,said dividing member may comprise a valve or a filter.

For receiving the treated biological material a container is provided,which is at least connectable to an outlet opening of said chamber, forexample, connected to said chamber building one piece or connectable tosaid chamber via a connecting member, preferably a Luer lock. Apartition member may be disposed between said chamber and saidcontainer. Said partition member is preferably a valve or a filterelement. It is appropriate to choose the material of the filter elementsuch that the treated biological material can pass the filter whilebigger particles and complexes are retained. In this way, it can beavoided that objectionable components which were produced duringelectric treatment are present in the final product. This is primarilyimportant with clinical use.

In an advantageous embodiment of the invention, the container comprisesat least one wall area which is self-sealing and can be perforated,preferably by a canula. Alternatively, the container may be equippedwith at least one outlet comprising a connecting member, preferably aLuer lock. Both embodiments allow easy and aseptic removal of thetreated biological material from the container. Said container may alsobe, for example, a syringe or an infusion pot allowing the treatedbiologic material to be directly applied to a person to be treated, forexample, in clinical practice.

Container and chamber may form a unit which is aseptically sealed inrelation to the outside so as to be transported and stored in common.

The wall area of the chamber and/or the container, which is self-sealingand can be perforated comprises a synthetic material, for example apolysiloxane, an elastomer or rubber or a foil made of plastic. The foilcould be made, for example, of acetyl cellulose.

In a preferred embodiment of the invention, said chamber comprises twooppositely arranged electrodes which are each placed in contact withsaid inner space.

Alternatively, a further electrode can be introduced into said innerspace of said chamber.

Particularly preferred, said electrode or said electrodes comprise(s) anelectroconductive synthetic material, preferably a plastic materialwhich is doped with conductive material, so that no metal ions which aretoxic for living cells can be emitted from said electrodes. Thisbenefits the survival rate of cells when living cells, in particulareukaryotic cells, are treated.

According to the invention the above ploblem is solved by a method,wherein said biological material is almost completely rinsed out of saidinner space of said chamber by means of a solution after said electricfield is generated, said solution being guided via an inlet line of saidchamber along at least one electrode. Thus, biological material adheringto inner surfaces of the chamber, in particular the electrode regions,can be effectively removed leading to approximately complete recovery ofthe material employed.

It is thereby advantageous if said solution is guided along saidelectrode at high flow rate.

Since biological material, in particular living cells, preferablyadheres to the cathode, in a beneficial embodiment of the methodaccording to the invention the solution is at first guided along thecathode.

In a further embodiment of said method, the biological material isintroduced into said inner space of said chamber by means of a syringeor the like through a wall area which is self-sealing and can beperforated.

In an advantageous embodiment of said method, a separating unit isopened by extraneous mechanical impact, said separating unit separatingsaid inner space of said chamber from a reservoir which contains saidsolution, said reservoir being connected or connectable to said chambervia said inlet line. In this manner, solution and chamber may betransported and stored together without unmeant contamination of theinner space by said solution. Thus, the inner space can be selectivelyrinsed, advantageously under aseptic conditions. Additionally, thesolution may be introduced into the inner space shortly after electrictreatment so that a buffer solution that is adapted to electrictreatment but less suitable for the biological material can eventuallybe diluted by said solution. The separating unit may thereby be a valvewhich can be opened by extraneous mechanical impact at least in onedirection or a fragile membrane which can be destroyed by extraneouslyapplied pressure. The fragile membrane is preferably made of a syntheticmaterial, for example polyvinylene, polysterol, polyethylene oder foilsmade of cellulose. Thereby, the synthetic material may be coated withfluorohalocarbon which has a low permeability for water vapor and a goodmechanical destrucibility.

In a beneficial embodiment of said method, it is further provided thatbiological material and solution, respectively, are introduced into acontainer which is at least connectable to an outlet opening of saidchamber. Using this container the treated material can then directly beprovided for further use in a very simple manner.

In a further embodiment of the invention, a reservoir which containssaid solution is at least partially formed by an elastic or deformablewall and a pressure is extraneously applied to said wall. In thismanner, the solution is rinsed into the chamber under pressure leadingto further improvement of efficiency of said method. Furthermore, thepressure applied may advantageously result in opening of the separatingunit which separates the inner space of the chamber from the reservoirwhich contains the solution so as to easily break the separation underaseptic conditions.

The biological material may also be rinsed into said container through apartition member, in partiucular a valve or filter, which is disposedbetween said chamber and said container so as to accomplish rinsingselectively and/or with removal of perturbing components.

In particular in clinical practice it may be beneficial to remove thetreated biological material from said container using a syringe or thelike through a wall area which is self-sealing and can be perforated.Necessary sterility is hereby garanteed, and additionally simple anddirect use of the treated material is ensured.

In an alternative embodiment of said method, the biological materialcomprises living cells, preferably eukaryotic cells, derivatives ofcells, subcellular particles or vesicles, into which biologically activemolecules, preferably nucleic acids, are transferred by generation ofsaid electric field, or which are fused by generation of said electricfield.

Said biologically active molecules may already be solved in a buffersolution and introduced into the inner space of said chamber before thebiological material is added. This measure significantly facilitates themethod since merely biological material has to be added by the user.

In one embodiment of the invention, the transfer of said biologicallyactive molecules into said living cells is achieved by a current densityof up to 120 A/cm², preferably 80 A/cm², or by a voltage pulse having afield strength of 2-10 kV*cm⁻¹ and a duration of 10-200 μs.

In a further embodiment of the invention, the transfer of saidbiologically active molecules into said living cells is achieved by acurrent flow following said voltage pulse without interruption, saidcurrent flow having a current density of 2-14 A/cm², preferably 5 A/cm²,and and a duration of 1-100 ms, preferably 50 ms.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further explained in detail by example of thefigures.

FIG. 1 shows lateral views, partially perspective and partiallysectional views, respectively, of a special embodiment of the deviceaccording to the invention in respect of different steps of the methodaccording to the invention;

FIG. 2 shows two sectional views of a further embodiment of the deviceaccording to the invention, which are rotated 90° in relation to eachother;

FIG. 3 shows a front sectional view of a further embodiment of theinvention;

FIG. 4 shows a lateral sectional view of the embodiment according toFIG. 3;

FIG. 5 shows a front sectional view of an additional alternativeembodiment of the device according to the invention;

FIG. 6 shows a sectional view of a part of the device according to theinvention, including a serpent-like chamber;

FIG. 7 shows a schematic representation of an embodiment of theinvention having a u-shaped chamber; and

FIG. 8 shows a schematic representation of a further embodiment of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows lateral views, partially perspective and partiallysectional views, respectively, of a special embodiment of the deviceaccording to the invention. In this particularly advantageous embodimentthe device according to the invention is a unit which is asepticallysealed and water-proof in all directions, and which allows treatment ofcells under perfect aseptic conditions.

FIG. 1 a shows a sectional view of the bottom region of the deviceaccording to the invention. This region of said device has a chamber 1which comprises an inner space 2 that serves for receiving biologicalmaterial, for example a suspension of living cells. Two coplanarelectrodes 3, 4 are inserted in this inner space 2, by which the innerspace 2 is bordered at two lateral areas in the bottem region of thechamber 1. In the upper region, the chamber 1 comprises a filter 5 whichis depicted merely in part in this figure. The filter 5 separates theinner space 2 from another part of the device. The inner space 2 mayhold, for example, a buffer solution 6 which may comprise biologicallyactive molecules, e.g. nucleic acids. For example, for use in genetherapy molecules solved in said buffer solution 6 may already bepresent within the chamber 1. A user merely has to introduce cells whichhave been recovered from a patient into the inner space 2 of saidchamber 1 and then transfer DNA into said cells by generating anelectric field.

FIG. 1 b shows a lateral perspective view of the device according to theinvention as depicted in FIG. 1 a, wherein the device is rotated 90° inrelation to FIG. 1 a. In this representation, the electrode 3 covers theview to the bottom region of the inner space 2. It becomes apparent fromthis representation that the chamber 1 comprises an inlet line 7 havingan opening 8 which is disposed in direct vicinity of the electrodes 3,4. The chamber 1 is connected to a reservoir 9 in one piece, whereinsaid reservoir 9 contains a solution 10 for rinsing the inner space 2 ofsaid chamber 1. The chamber 1 and the reservoir 9 are separated fromeach other by a separating unit 11. This separating unit 11 may be, forexample, a valve-like lock or a fragile membrane. Additionally, thechamber 1 is linked to a container 12 in one piece, said container 12being separated from the inner space 2 of said chamber 1 by the filter5. Said container 12 and said reservoir 9 each form a unit with saidchamber 1, which is aseptically sealed and water-proof in relation tothe outside. The chamber 12 further comprises a wall area 13 that can beperforated and through which the canula 14 of a syringe 15 may beinserted into the inner sopace 2. The syringe 15 may contain, forexample, a suspension 16 of living cells, which can be injected into theinner space 2 through the canula 14. The suspension of cells is pressedinto the inner space 2 by application of pressure in direction of arrow22, and is hence available for the following electric treatment. Thecells may be, for example, primary cells taken from a patient, whichshall be transfected with suitable DNA for gene therapy. A suitableelectric field is then generated within the inner space 2 by applicationof voltage to the electrodes 3, 4, by which DNA can be efficientlytransferred into the cells, in particular directly into the nucleus.

FIG. 1 c shows the device according to the invention as depicted in FIG.1 b when the inner space 2 of chamber 1 is rinsed after electricallytreating the biological material. It becomes apparent from thisrepresentation that the wall 17 of the reservoir 9 is made of an elasticmaterial which can be deformed, for example an elastic plastic material.The wall 17 is deformed in direction of arrow 18 by application ofpressure on wall 17 and thus pressure is applied to the solution 10within the reservoir 9. Die to this pressure separating unit 11 betweenreservoir 9 and chamber 1 can be opened. Alternatively, the separatingunit 11 may be opened by another manipulation from the outside. Thus,solution 10 reaches the inner space 2 of chamber 1 via inlet line 7.Since the opening 8 of inlet line 7 is disposed near (in vicinity of)electrodes 3, 4, in particular these electrodes 3, 4 are intensivelyrinsed by solution 10 with high flow rate. Hereby, the flow rate can bespecifically increased by the pressure applied to wall 17. In order toavoid dead volume within inner space 2 the inner surface of chamber wall19 is designed with rounded nooks so as to allow optimal rinse if innerspace 2. Due to this measure treated cells can be suspended almostcompletely in solution 10. By disposing the opening 8 near bothelectrodes 3, 4 these are rinsed evenly, wherein turbulances areproduced if the distance between both electrodes is small, which arebenficial for the rinsing procedure. Alternatively, the opening may bedisposed closer to one electrode which is preferably the cathode. Due tothe particularly advantageous construction of the device according tothe invention cells adhered to walls and electrodes can be recovered,wherein suspension of cells is ensured even when foam formation occursif high field strengths are used.

FIG. 1 d shows the device according to the invention as depicted in FIG.1 b after the inner space 2 of chamber 1 has been rinsed by solution 10.Rotating the device 180° the solution 10 including the treated cells canreach the container 12 through filter 5. This state is depicted in FIG.1 e. Alternatively, the device according to the invention may already berotated 180° prior rinsing, wherein a slightly higher pressure has to beapplied to solution 10 so as to be pressed at first into the inner space2 of chamber 1 contrary to gravity. Using the filter 5 larger particlescan be retained in the inner space 2 so as to avoid perturbing effectson the treated suspension of cells. As chamber 1 also container 12 has aperforatable wall area 20 through which the suspension of cells may beremoved out of said container 12 by means of a syringe 21. Thefunnel-like shape of the container 12 is thereby beneficial because thecells sedimenting by-and-by are concentrated in the bottom region andthus can be recovered concentrated as well. Accordingly, a sterilesuspension of treated cells can be applied to a patient fast,concentrated and in an easy manner.

FIG. 2 shows two sectional views of a further embodiment of the deviceaccording to the invention, which are rotated 90° in relation to eachother. The device shown comprises a chamber 25 having an inner space 26which contains a suspension 27 of cells and/or solved biologicallyactive molecules. The chamber 25 further includes two flat electrodes28, 29 having a plane surface, which are arranged parallel to each otherat opposing side walls of said chamber 25. In the representation of FIG.2 a only electrode 28 which is disposed behind the cut surface isvisible. The chamber 25 has two connecting members 30, 31 which aredisposed at opposing ends of chamber 25. The introduction of cellsuspension 27 is accomplished through connecting member 31, preferablyfrom a container having a canula which has a length that corresponds tothe thickness of connecting member 31 so that there is no dead volume inthe inner space 26 when cells are introduced. The canula may pierce amembrane or the like by which the inner space 26 is sealed in relationto the outside. A reservoir 32 can be connected to connecting member 30,wherein said reservoir 32 may be, for example, a syringe or the like.The reservoir 32 may be elastic and flexible so that its wall can bedeformed when, in an alternative embodiment, the solution is sucked outof said reservoir 32 by negative pressure generated by container 40within inner space 26. The canula 33 of the reservoir 32 can be insertedinto a recess 34 of connecting member 30. Said recess 34 may be sealed,for example, by a plug or closed at the passage to inlet line 36 by avalve or a fragile membrane which can be pierced by canula 33. Thus,solution 35 can be transferred from reservoir 32 into inlet line 36 ofchamber 25. Said solution 35 reaches the inner space 26 through opening37 of inlet line 36, wherein electrode 29 is intensely rinsed by saidsolution 35. Advantageously, electrode 29 is the cathode on which cellmaterial primarily adheres while electroporating cells, which is hardlyremovable with common devices. As it becomes apparent from FIG. 2 a theinner surface 38 of chamber wall 39 is rounded so as to avoid deadvolume. In this manner, it is ensured that inner space 26 can beeffectively rinsed. Subsequently, the suspension 27 of cells, which isdiluted by solution 35, is received by the container 40 so that, in oneembodiment, rinsing and removing of cells is accomplished in two steps.Alternatively, the solution can be sucked out of the reservoir 32through inner space 26 along the electrodes 28, 29 into the container 40by negative pressure generated by said container 40 within inner space26 so that here rinsing and removing is accomplished in one step. Thecanula 41 of the container 40 which may be, for example, a syringe orthe like is inserted into a recess 42 of the connecting member 31, aswith connecting member 30. Said connecting members 30, 31 may be, forexample, Luer locks which can be conncted with syringes or infusion potsin commonly known manner. But said connecting members 30, 31 may alsobe, for example, rubber plugs which can be perforated.

FIG. 3 shows a front sectional view of a further embodiment of theinvention. The device depicted in this representation comprises achamber 45 including an inner space 46 and two coplanar electrodes 47,48. This chamber 45 further includes two parallel inlet lines 49, 50which are disposed in the side areas of chamber 45. Through said inletlines 49, 50 a solution for rinsing the inner space 46 may beintroduced. But said inlet lines 49, 50 may also be used, for example,to introduce the biological material into the inner space 46 and,subsequently, to pour in said solution. The inlet lines are fed via atube-like overflow, i.e. the oblong overflow channel 59, what isdescribed in detail with respect to FIG. 4. An outlet opening 51 isdisposed between both inlet lines 49, 50, through which the content ofthe inner space 46 of chamber 45 can be recovered. Hereby, the contentof said chamber flows into an oblong drain pipeline 60 which isdescribed in detail with respect to FIG. 4. Referring to FIG. 3, apartition member 61 is disposed between the inner space 46 and the drainpipeline 60, which may be, for example, a filter membrane.

FIG. 4 shows a lateral sectional view of the embodiment according toFIG. 3. Here it becomes apparent that chamber 45 is designedlongitudinally extended so as to be suitable for receiving largervolumes. Electrode 48 as well as electrode 47 which is not visible inthis representation extend along the entire length of the chamber 45,both electrodes 47, 48 having a minor hight. In the inlet area theoverflow channel 59 can be conncted to a reservoir and a solution isguided through said overflow channel 59 from said reservoir into theinlet lines which are not visible in this representation (referencenumbers 49 und 50 in FIG. 3). Said inlet lines extend seam-like alongthe entire length of the chamber 45 as well. The inlet lines have asmaller cross-section than the overflow channel 59 so that a constantpressure along the entire length of the chamber 45 is produced if theoverflow channel 59 is filled with solution. In the outlet area, thedrain pipeline 60 which extends parallel to the overflow channel 59 canbe connected to a container which receives the treated biologicalmaterial rinsed out by the solution.

FIG. 5 shows a further alternative embodiment of the invention, whichgenerally corresponds to the device according to FIGS. 3 and 4, whereinin this embodiment only one inlet line 52 is provided. Here, the opening53 is disposed in direct vicinity of electrode 54 which is preferablythe cathode. The inlet line 52 is fed with solution by channel 62,wherein the solution comprising the treated material can be recoveredthrough partition member 64 and channel 62, respectively.

FIG. 6 a sectional view of a part of the device according to theinvention, including a serpent-like chamber 55. Electrodes are notdepicted in this representation but may be disposed in front and behindthe cut surface. The chamber 55 includes a tube-like inlet line 56 and atube-like drain 57 which each are disposed at the ends of theserpent-like inner space 58. Inlet line 56 und inner space 58,respectively, each have an even minor cross-section leading to high flowrate in the inner space 58 and at the surfaces of the electrodes whichare not depicted in this representation. Thus, the chamber can be rinsedvery effectively and completely. The inner cross-section of inlet line56 may narrow in direction of inner space 58 so as to increase the flowrate of the solution within said inner space 58.

FIG. 7 shows a further embodiment of the invention having a u-shapedchamber 65. In this embodiment, the electrodes which are not depicted inthis representation are disposed in front and behind the cut surface.The solution is introduced into the inner space 66 via inlet line 67,wherein a high flow rate can be achieved due to the minor cross-sectionof chamber 65. The solution comprising the treated biological materialis pressed or sucked out of chamber 65 via drain pipeline 68.

FIG. 8 shows an alternative embodiment of the invention, which generallycorresponds to the embodiment according to FIG. 7. However, in thisembodiment the inlet line 76 of the chamber 75 has a significantlylarger cross-section than the drain pipeline 77 so that larger volumescan be processed.

LIST OF REFERENCE NUMBERS

-   1 Chamber-   2 Inner space-   3 Electrode-   4 Electrode-   5 Filter-   6 Buffer solution-   7 Inlet line-   8 Opening-   9 Reservoir-   10 Solution-   11 Separating unit-   12 Container-   13 Wall area-   14 Canula-   15 Syringe-   16 Suspension-   17 Wall-   18 Arrow-   19 Chamber wall-   20 Wall area-   21 Syringe-   22 Arrow-   25 Chamber-   26 Inner space-   27 Suspension-   28 Electrode-   29 Electrode-   30 Connecting member-   31 Connecting member-   32 Reservoir-   33 Canula-   34 Recess-   35 Solution-   36 Inlet line-   37 Opening-   38 Surface-   39 Chamber wall-   40 Container-   41 Canula-   42 Recess-   45 Chamber-   46 Inner space-   47 Electrode-   48 Electrode-   49 Inlet line-   50 Inlet line-   51 Outlet opening-   52 Inlet line-   53 Opening-   54 Electrode-   55 Chamber-   56 Inlet line-   57 Drain-   58 Inner space-   59 Overflow channel-   60 Drain pipeline-   61 Partition member-   62 Channel-   63 Channel-   64 Partition member-   65 Chamber-   66 Inner space-   67 Inlet line-   68 Drain pipeline-   75 Chamber-   76 Inlet line-   77 Drain pipeline

1-37. (canceled)
 38. Device for treating biological material comprisingat least one chamber which at least can be closed to the outside, saidchamber comprising an inner space for receiving said biologicalmaterial, at least one electrode for generating an electric field andwhich is in contact with said inner space of said chamber, at least oneinlet line which comprises at least one opening which is disposed atsaid electrode, wherein at least one reservoir for receiving a solution,which is formed by a wall, is connectable or connected to said innerspace via said inlet line, and wherein said inner space of said chamberand said reservoir are separated from each other by a separating unitwhich is designed so that a separation created by said separation unitcan be broken by extraneous mechanical impact.
 39. The device of claim38, wherein said inlet line is tube-like.
 40. The device of claim 38,wherein the inner diameter of said inlet line decreases in the directionof said electrode.
 41. The device of claim 38, wherein said separatingunit is a valve or a fragile membrane which can be destroyed by applyingpressure.
 42. The device of claim 38, wherein said chamber is at leastaseptically sealed to the outside.
 43. The device of claim 38, whereinsaid wall forming said reservoir comprises an elastic and/or deformablematerial.
 44. The device of claim 38, wherein said reservoir is at leastconnected to said chamber forming one piece with the chamber orconnectable to said chamber via a connecting member.
 45. (canceled) 46.The device of claim 44, wherein said chamber and said reservoir form aunit which is at least aseptically sealed to the outside.
 47. The deviceof claim 38, wherein said chamber comprises at least one wall area whichis self-sealing and can be perforated or which is equipped with at leastone inlet comprising a connecting member.
 48. (canceled)
 49. The deviceof claim 38, wherein said chamber is formed like a serpent and/orspiral.
 50. The device of claim 38, wherein said chamber is divided intoseveral subunits by at least one dividing member.
 51. The device ofclaim 50, wherein said dividing member comprises a valve and/or afilter.
 52. The device of claim 38 further comprising a container,wherein said container is at least connectable to or connected to anoutlet opening of said chamber or connectable to said chamber via aconnecting member.
 53. The device of claim 52, wherein said container isconnected to said chamber forming one piece with said chamber. 54.(canceled)
 55. The device of claim 52, wherein a partition member isdisposed between said chamber and said container.
 56. The device ofclaim 55, wherein said partition member is a valve or a filter.
 57. Thedevice of claim 52, wherein said container comprises at least one wallarea which is self-sealing and can be perforated or which is equippedwith at least one outlet comprising a connecting member.
 58. (canceled)59. (canceled)
 60. The device of claim 52, wherein said container andsaid chamber form a unit which is aseptically sealed to the outside. 61.The device of claim 47, wherein said wall area which is self-sealing andcan be perforated comprises a synthetic material.
 62. (canceled)
 63. Thedevice of claim 38, wherein said chamber comprises two oppositelyarranged electrodes which are in contact with said inner space, orwherein a further electrode can be introduced into said inner space ofsaid chamber.
 64. The device of claim 38, wherein said electrode orelectrodes comprise(s) an electro-conductive synthetic material.
 65. Thedevice of claim 64, wherein said electro-conductive synthetic materialis a plastic material which is doped with conductive material.
 66. Amethod for treating biological material comprising: providing an innerspace of a chamber which at least can be closed to the outside, saidinner space comprising at least one electrode which is placed in contactwith said inner space of said chamber for generating an electric fieldin said inner space after introducing said biological material byapplying voltage to said electrode and a further electrode which is incontact with said inner space of said chamber, introducing saidbiological material into said inner space of said chamber, aftergenerating the electric field, almost completely rinsing said biologicalmaterial out of said inner space of said chamber with a solution, saidsolution being guided from a reservoir containing said solution via aninlet line of said chamber along at least one electrode and saidreservoir being connected or connectable to said chamber via said inletline, and wherein a separating unit which separates said inner space ofsaid chamber from said reservoir is opened by extraneous mechanicalimpact.
 67. The method of claim 66, wherein said solution is guidedalong said electrode under pressure.
 68. (canceled)
 69. The method ofclaim 66, wherein said biological material is introduced into said innerspace of said chamber with a syringe or a syringe-like device through awall area which is self-sealing and can be perforated.
 70. The method ofclaim 66, wherein said separating unit is a valve which can be opened byextraneous mechanical impact at least in one direction, or a fragilemembrane which can be destroyed by extraneously applied pressure. 71.The method of claim 66, wherein said biological material and saidsolution, respectively, are introduced into a container which is atleast connectable to an outlet opening of said chamber.
 72. The methodof claim 66, wherein said reservoir which contains said solution is atleast partially formed by an elastic and/or deformable wall and apressure is extraneously applied to said wall.
 73. The method of claim66, wherein said biological material is rinsed into said containerthrough a partition member, which is disposed between said chamber andsaid container.
 74. The method of claim 73, wherein said partitionmember is a valve and/or filter.
 75. The method of claim 70, whereintreated biological material is removed from said container using asyringe or syringe-like device through a wall area which is self-sealingand can be perforated.
 76. The method claim 66, wherein said biologicalmaterial comprises living cells, derivatives of cells, sub-cellularparticles and/or vesicles, into which biologically active molecules aretransferred by generation of said electric field, or which are fused bygeneration of said electric field.
 77. (canceled)
 78. (canceled)
 79. Themethod of claim 76, wherein said biologically active molecules aredissolved in a buffer solution and introduced into the inner space ofsaid chamber before the biological material is added.
 80. The method ofclaim 76, wherein the transfer of said biologically active moleculesinto said living cells is achieved via a current density of up to 120A/cm², preferably 80 A/cm², or by a voltage pulse having a fieldstrength of 2-10 kV*cm⁻¹ and a duration of 10-200 μs.
 81. The method ofclaim 80, wherein the transfer of said biologically active moleculesinto said living cells is achieved by a current flow following saidvoltage pulse without interruption, said current flow having a currentdensity of 2-14 A/cm² preferably 5 A/cm ², and a duration of 1-100 ms,preferably 50 ms.